Robustanic acid as a glutaminase and Na+, K+-ATPase inhibitor from leaves of Eucalyptus globulus.

This study was to compare glutaminase and Na+, K+-ATPase inhibitory activities of 20 herbal extracts and investigate the isolation, structural elucidation and those inhibitory activities of three triterpenes from the selected extract of Eucalyptus globulus Labill. Three triterpenes, ursolic acid (1), robustanic acid (2) and ursolic acid lactone (3), were identified by analyzing their NMR and MS spectral data and comparison of these with reported data. The IC50 values of 1-3 and the control compound against glutaminase, 6-diazo-5-oxo-l-norleucine (DON), were 443 µM, 334 µM, 963 µM and 134 µM, respectively. The IC50 values of 1, 2 and the control compound against Na+, K+-ATPase and ouabain, were 180 µM, 56 µM and 0.5 µM, respectively. Compounds 1 and 2 may serve as potential lead compounds for the prevention and treatment of neurodegenerative and lifestyle-related diseases by targeting glutaminase and Na+, K+-ATPase. This is the first report on glutaminase and Na+, K+-ATPase inhibitory activities of 2.

Photostability evaluation of manidipine tablets and structural determination of its photoproducts.

Manidipine (MP) is a dihydropyridine drug, which is treated for the reduction of high blood pressure. The aim of this study is to clarify the photochemical behavior of MP in the case of ultraviolet light (UV) irradiation for MP tablets (Calslot® tablets). The tablets and its altered forms (powders and suspensions) were UV-irradiated using a black light, and residual amounts of active pharmaceutical ingredients (APIs) were monitored by high-performance liquid chromatography (HPLC). Due to the photoproducts of MP were detected in HPLC chromatograms, the elucidation of their chemical structures was carried out utilizing electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). As a result, APIs in Calslot® tablets were almost completely photodegraded in the case that Calslot® tablets were suspended in an aqueous media along with the generation of some MP photoproducts. LC-ESI-MS/MS analysis clarified the chemical structures of three MP photoproducts, indicating that they were a pyridine analogue, benzophenone and a hydrolysate. Benzophenone was a main MP photoproduct. It was possible that MP might be firstly oxidized to form its pyridine analogue, followed by the oxidation of a dimethyl methylene moiety. This moiety seemed to be eliminated as a benzophenone, and the cleavage of an ester bond of the residual moiety resulted in the generation of a hydrolysate. Finally, toxicological potencies of MP and its photoproducts were predicted in silico toxicity evaluation, suggesting some of biological effects of the photoproducts might be altered compared with MP.

The evaluation of photochemical behavior of antihistaminic drug triprolidine in an aqueous media.

Photodegradation is widely known as a changer of both the quality and the quantity of several chemical compounds. In this study, we sought to examine the photochemical behavior of triprolidine (TRP), which is a member of the first-generation antihistamine and utilized for a relief of an allergic conditions, in an aqueous media. There are no reports focused on its potential photoproducts and photodegradation pathways in detail to the best of our knowledge. TRP photodegradation induced by ultraviolet light (UV) irradiation was monitored utilizing high-performance liquid chromatography (HPLC), and structural elucidation of the TRP photoproduct was performed by electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR). Finally, the mechanism of TRP photodegradation was speculated based on the identified photoproduct. TRP was photodegraded dependent on the irradiation time of UV in proportion to the generation of one TRP photoproduct (TRP-P). Structural determination by LC-ESI-MS/MS and NMR clarified that TRP-P was the geometrical isomer of TRP, which was formed by the cis-trans conversion of double bond. UV irradiation experiment for TRP-P revealed the conversion from it to TRP. It is clarified that cis-trans conversion between TRP and TRP-P is photo-equilibrium reaction and TRP-P is predominant under the condition as this experiment. Toxicological potencies of TRP and TRP-P might not be observed by ProTox-II in silico toxicity evaluation. This is the first study evaluating the photochemical behavior of TRP.

GABase and glutaminase inhibitory activities of herbal extracts and acylated flavonol monoglycosides isolated from the leaves of Laurus nobilis L.

This study was to compare GABase [a mixture of γ-aminobutyric acid (GABA) aminotransferase and succinic semialdehyde dehydrogenase] and glutaminase inhibitory activities of 20 herbal extracts and investigate the isolation, structural elucidation and those inhibitory activities of three acylated flavonol monoglycosides from the selected extract of Laurus nobilis L. (laurel). On the basis of the NMR spectroscopic data and the ESI MS spectra together with the comparison with the literature values, three compounds were identified as kaempferol-3-O-(4″-E-p-coumaroyl)-α-l-rhamnopyranoside (1), kaempferol-3-O-(3″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside (2) and kaempferol-3-O-(2″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside (3), respectively. The IC50 values of GABase inhibitory activity of 1-3 and p-hydroxybenzaldehyde (HBA) as control were 0.24 mM, 0.14 mM, 0.12 mM and 0.43 mM, respectively. Additionally, the IC50 values of glutaminase inhibitory activity of 1-3 and 6-diazo-5-oxo-l-norleucine (DON) as control were 0.34 mM, 0.13 mM, 0.14 mM and 0.33 mM, respectively. The results suggest that the extract from laurel shows the strongest biological activities among 20 herbal extracts and three acylated flavonol monoglycosides may serve as potential lead compounds for the prevention and treatment of neurodegenerative and lifestyle-related diseases by targeting GABase and glutaminase. This is the first report on GABase and glutaminase inhibitory activities of 1-3.

Pentacyclic triterpene acids, rotungenic acid and barbinervic acid, from fresh leaves of Diospyros kaki Thunberg and their glutaminase inhibitory activities.

Glutaminase is an important target that is often over-expressed in neurodegenerative and lifestyle-related diseases but few effective inhibitors of this enzyme have yet reached clinical trials. Three compounds isolated from fresh leaves of Diospyros kaki Thunberg, ursolic acid (1), rotungenic acid (2) and barbinervic acid (3), were identified by analyzing their NMR and MS spectral data and comparison of these with reported data. The IC50 values of 1-3 and 6-diazo-5-oxo-L-norleucine (DON) as control were 775, 13, 14, and 434 µM, respectively. Compounds 2 and 3 showed higher glutaminase inhibitory activities than DON. Compounds 2 and 3 may serve as potential lead compounds for the prevention and treatment of neurodegenerative and lifestyle-related diseases by targeting glutaminase. This is the first report on glutaminase inhibitory activities of 2 and 3.

Omphalines A-E: ent-Rosane-Type Diterpenoids from the Madagascar Endemic Plant, Omphalea oppositifolia.

From the less polar fraction of the MeOH extract of the leaves and twigs of Omphalea oppositifolia, five new ent-rosane-type diterpenoids, named omphalines A-E (1-5), were isolated together with one known compound, 7-keto-ent-kaurane-16ß,17-diol (6), by a combination of various kinds of chromatography. The structure of omphaline A (1) was elucidated to be 19-nor-ent-rosane-4,15-diene-2ß,6α-diol-3-one. Omphalines B (2), C (3), D (4), and E (5) possessed two double bonds at 5- and 15-positions, and hydroxy functional groups at 3ß-, 2α,3α-, 2α,3ß-, and 2α,19-positions, respectively. The absolute configuration of 1 was determined by the comparison of the experimental electronic circular dichroism (ECD) spectrum and calculated ECD spectra.

Crotofolane-Type Diterpenoids: Crotocascarins R-V, Rearranged Trinorcrotofolane: Crotocascarin δ and a Phorbol Derivative from the Leaves of Croton cascarilloides.

Five new crotofolanes, named crotocascarins R-V (1-5), one rearranged trinorcrotofolane, crotocascarin δ, and one phorbol derivative were isolated from the EtOAc-soluble fraction of the MeOH extract of the leaves of Croton cascarilloides. Crotocascarins R (1), T (3), and U (4) possessed isobutyric acid as an acyl moiety and crotocascarin B (2) an acetyl group, whereas crotocascarin V (5) was elucidated to be a hydroxylated compound of crotocascarin K at the 9-position. Crotocascarin δ (6) was a trinor rearranged crotofolane with a tertiary hemiketal functional group at the 8-position. The absolute configuration of the 8-position was determined by the comparison of the experimental electronic circular dichroism (ECD) spectrum and calculated ECD spectra. Compound 7 was a phorbol ester derivative with a peroxide functional group. The fatty acid attached at the 12-position was found to be a single species-i.e., lauric acid (C-12)-from the evidence of the mass spectral data.

Glutaminase inhibitory activity of umbelliferone isolated from kabosu (Citrus sphaerocarpa Hort. ex Tanaka).

Kabosu (Citrus sphaerocarpa Hort. ex Tanaka) fruits have pleasant and fresh odors and have been used as raw materials for vinegar, seasonings, jams, marmalades and juices in Japan. The n-butanol extracts from kabosu fruits were prepared and a component in the extract was purified by column chromatography and HPLC to afford compounds 1-3. Three compounds, 5-(hydroxymethyl)-2-furaldehyde (1), umbelliferone (2) and oxypeucedanin hydrate (3), have been isolated from kabosu, and the structures of compounds 1-3 were elucidated by 1 D and 2 D NMR as well as EI-MS. Compound 2 exhibited potent glutaminase inhibitory activity with an IC50 value of 1.33 mM. This is the first report on glutaminase inhibitory activity of 2 and the isolation of three compounds 1-3 from kabosu fruits.

Glutaminase inhibitory activities of pentacyclic triterpenes isolated from Thymus vulgaris L.

Glutaminase is an important target that is often over expressed in neurodegenerative and　lifestyle related diseases but few effective inhibitors of this enzyme have yet reached clinical trials. Ursolic acid (1), betulinic acid (2) and oleanolic acid (3), three pentacyclic triterpene acids, have been isolated from the leaves of Thymus vulgaris L. Enzyme inhibition experiments demonstrated their inhibitory effects against glutaminase activity. Compound 2 significantly inhibited the glutaminase activity with IC50 of 0.31 mM, stronger than the positive control 6-diazo-5-oxo-L-norleucine (DON) with IC50 of 0.57 mM. Compound 2 may serve as a potential lead compound for the prevention and treatment of neurodegenerative diseases and lifestyle related diseases by targeting glutaminase. This is the first report on glutaminase inhibitory activities of 1-3 isolated from T. vulgaris L.

Detecting and Differentiating Monosaccharide Enantiomers by 1H NMR Spectroscopy.

Monosaccharides play important roles in living organisms. They are present in essential glycoproteins, nucleic acids, and glycolipids as well as cell walls and bioactive natural product glycosides and polysaccharides. Monosaccharides are optically active, and as a routine, scientists make sure that their absolute configurations are determined when new natural glycosides are isolated. Many determination methods for the absolute configuration of monosaccharides have been reported, and thus far, taking advantage of their optical rotation differences is the most used and efficient method to distinguish enantiomers. This method, however, is not very convenient, because it requires a milligram amount of each pure sample and the availability of a polarimeter. Identification methods dealing with comparison of the retention times of the d- and l-diastereomeric monosaccharide derivatives by GC, TLC Rf values, HPLC, or UPLC have been also reported. Although effective, these methods still require sample preparation and a few milligrams of the test compounds. A new method with simple sample preparation to distinguish enantiomers of monosaccharides by analyzing the 1H NMR spectra of their diastereomeric derivatives has been developed. The monosaccharide components of a commercially available saponin-rich Panax ginseng and monoglycosides have been successfully identified using this procedure.

Ardisiatetrons A and B: tetronic acid derivatives and triterpenes from the leaves of Ardisia quinquegona and their biological activity.

From the leaves of Ardisia quinquegona, two alkylated tetronic acid derivatives, named ardisiatetrons A and B (1, 2), and four triterpenoids (3-6) were isolated together with one known compound (7) by a combination of various kinds of chromatography. The structure of new methyl migrated triterpene (3) was confirmed by X-ray crystallographic analysis. Compounds 2, 3, and 7 showed moderate anti-Leishmania activity and cytotoxicity towards A549 cells.

Photoprotective Effects of Selected Amino Acids on Naproxen Photodegradation in Aqueous Media.

It is important to develop a photostabilization strategy to ensure the quality of photosensitive compounds, including pharmaceuticals. This study focused on the protective effects of 20 amino acids on the photodegradation of naproxen (NX), a photosensitive pharmaceutical, to clarify the important nature of a good photostabilizer. Our previous report indicated the photodegradability of NX and the protective effects of some antioxidants on its photodegradation, therefore, this compound was used as a model compound. The degradation of NX in aqueous media during ultraviolet light (UV) irradiation and the protective effects of selected amino acids were monitored through high-performance liquid chromatography (HPLC), equipped with a reverse-phase column. Addition of cysteine, tryptophan, and tyrosine induced the significant suppression of NX photodegradation after UV irradiation for 3 h (residual amount of NX; 15.35%, 6.82%, and 15.64%, respectively). Evaluation of the antioxidative activity and UV absorption spectrum showed that cysteine suppressed NX degradation through its antioxidative ability, while tryptophan and tyrosine suppressed it through their UV filtering ability. Furthermore, three amino acids at higher concentrations (more than 100 µmol/L) showed more protective effects on NX photodegradation. For 10 mmol/L, residual amounts of NX with cysteine, tryptophan, and tyrosine were 58.51%, 69.34%, and 82.40%, respectively. These results showed the importance of both photoprotective potencies (antioxidative potency and UV filtering potency) and stability to UV irradiation for a good photostabilizer of photosensitive pharmaceuticals.

Comparative studies of the biological activities of selected herbal extracts and phenolic compounds isolated from Rosa gallica.

This study aimed to compare the biological activities of 35 herbal hydroethanolic extracts and select high potential extract, which showed antioxidative activity and inhibitory activities of α-glucosidase, lipase, and hyaluronidase, and to investigate the isolation, structural elucidation, and biological activities of five phenolic compounds from the selected extracts of Rosa gallica. On the basis of one-dimensional nuclear magnetic resonance together with the comparison with the literature values, the phenolic compounds were identified as methyl gallate (1), kaempferol-3-O-arabinofuranoside (2), multinoside A acetate (3), kaempferol (4), and quercetin (5), respectively. The results suggest that the extracts from R. gallica show the strongest biological activities in 35 herbal extracts and that 1, 4, and 5 among the five isolated compounds from rose extracts are effective in promoting antioxidative and enzymatic inhibitory activities.

Ebenamariosides A-D: Triterpene Glucosides and Megastigmanes from the Leaves of Diospyros maritima.

The 1-BuOH-soluble fraction of the methanol (MeOH) extract of Diospyros maritima was separated by chromatographic techniques to give three new oleanane-type and one new ursane-type triterpene glucoside, named ebenamariosides A-D (1-4); two megastigmanes were also isolated. The structures of triterpene glucosides was elucidated with extensive investigation by one and two dimensional NMR spectroscopy and the structures were confirmed by partial enzymatic hydrolyses to give the corresponding mono-glucosides and aglycones. The structures of the megastigmanes, including their absolute stereochemistries, were elucidated by spectroscopic evidence and by the modified Mosher's method. Two megastigmanes were chemically correlated and their absolute structures were unambiguously determined. The cytotoxicity of the triterpene glucosides and their degradation products were assayed. They did not show any significant activity.

Protective Effect of Selected Antioxidants on Naproxen Photodegradation in Aqueous Media.

A photostabilization strategy is an important aspect of quality assurance for photosensitive compounds. This study focused on the photoprotective effects of selected antioxidants including the effect of L-ascorbic acid (AA) on naproxen (NX) photodegradation in aqueous media. NX degradation during ultraviolet light (UV) irradiation and the protective effects of selected antioxidants were monitored by high-performance liquid chromatography (HPLC). The addition of AA induced the suppression of NX photodegradation, although the protective effect disappeared after AA was degraded completely. The results of the evaluations on the photoprotective effects on NX photodegradation and antioxidative activities of AA and other antioxidants showed that the protective effects of antioxidants are dependent on reducing power and photostability under UV irradiation. In this experiment, quercetin (QU) is the most effective antioxidant on account of the residual rate of QU after UV irradiation and the antioxidative activity in the potential antioxidant (PAO) test was significantly higher compared to other antioxidants following the higher protective effect on NX photodegradation.

Eight ent-Kaurane Diterpenoid Glycosides Named Diosmariosides A-H from the Leaves of Diospyros maritima and Their Cytotoxic Activity.

From the leaves of Diospyros maritima, collected from Okinawa Island, eight new glycosides based on ent-kaurane-type diterpenoids, entitled diosmariosides A-H, were isolated. The absolute structure of diosmarioside E (5) was determined by X-ray crystallographic analysis. The structure of diosmarioside H was elucidated to be a dimeric compound between diosmarioside A and a sugeroside through a ketal bond. An assay of cytotoxicity towards the lung adenocarcinoma (A549) cell line was performed. Among the compounds isolated, only diosmarioside D (4) and sugeroside 9 showed strong activity. The anti-microbial activity toward multi-drug resistant strains was also determined, but no activity was observed.

Halogenated Compounds from Directed Fermentation of Penicillium concentricum, an Endophytic Fungus of the Liverwort Trichocolea tomentella.

One new chlorinated xanthone, 6-chloro-3,8-dihydroxy-1-methylxanthone (1), a new 2-bromo-gentisyl alcohol (2), and a mixture of 6-epimers of 6-dehydroxy-6-bromogabosine C (3a and 3b), together with 19 previously identified compounds, epoxydon (4), norlichexanthone (5), 2-chlorogentisyl alcohol (6), hydroxychlorogentisyl quinone (7), 6-dehydroxy-6α-chlorogabosine C (8a), 6-dehydroxy-6ß-chlorogabosine C (8b), gentisyl alcohol (9), gentisyl quinone (10), (R,S)-1-phenyl-1,2-ethanediol (11), dehydrodechlorogriseofulvin (12), dechlorogriseofulvin (13), dehydrogriseofulvin (14), griseofulvin (15), ethylene glycol benzoate (16), alternariol (17), griseoxanthone C (18), drimiopsin H (19), griseophenone C (20), and griseophenone B (21), were isolated from cultures of Penicillium concentricum, a fungal endophyte of the liverwort Trichocolea tomentella. The structures of the new compounds (1, 2, 3a, and 3b) were elucidated by interpretation of spectroscopic data including one- and two-dimensional NMR techniques. Among these, compounds 2-4 displayed modest cytotoxicity to the MCF-7 hormone-dependent breast cancer cell line with IC50 values of 8.4, 9.7, and 5.7 µM, respectively, whereas compound 9 exhibited selective cytotoxicity against the HT-29 colon cancer cell line with an IC50 value of 6.4 µM. During this study we confirmed that the brominated gentisyl alcohol (2) was formed by chemical conversion of 4 during bromide salt addition to culture media.

Crotofolane-Type Diterpenoids, Crotocascarins L-Q, and a Rearranged Crotofolane-Type Diterpenoid, Neocrotocascarin, from the Stems of Croton cascarilloides.

From the stems of Croton cascarilloides, further crotofolane-type diterpenoids, named crotocascarins L-Q (1-6), and a rearranged one (7), named neocrotocascarin were isolated. Their structures were elucidated from spectroscopic evidence. Also, the structures of crotocascarin O (4) and neocrotocascarin (7) were determined by X-ray crystallographic analyses. As a result, neocrotocascarin (7) was found to possess a new carbon framework. A plausible mechanism for the formation of neocrotocascarin (7) is also discussed.

Crotocascarins I-K: Crotofolane-Type Diterpenoids, Crotocascarin γ, Isocrotofolane Glucoside and Phenolic Glycoside from the Leaves of Croton cascarilloides.

From the 1-BuOH-soluble fraction of a methanol (MeOH) extract of the leaves of Croton cascarilloides, crotofolanes: crotocascarins I-K, nor-crotofolane: crotocascarin γ, isocrotofolane glucoside and phenolic glycoside were isolated by a combination of various separation techniques. Their structures were elucidated mainly from the NMR spectroscopic evidence. The structure of crotocascarin K was first elucidated by spectroscopic analysis and then was confirmed by X-ray crystallographic analysis. Its absolute structure was finally determined by the modified Mosher's method. Isocrotofolane glucoside was found to possess a new skeleton, however, its absolute structure remains to be determined.

Nitrile-containing phenolic glucosides from the leaves of Glochidion acuminatum.

From the EtOAc-soluble fraction of a MeOH extract of the leaves of Glochidion acuminatum, six new compounds along with five known ones were isolated. The structures of the new compounds were elucidated to be two gallates, a p-hydroxybenzoate and an (S)-2-(4-hydroxycyclohex-1-en-1-yl)acetate of a nitrile-containing phenolic glucoside, methyl 2-(2-hydroxyphenyl)acetate ß-D-glucopyranoside, and (S)-methyl 2-[4-sulfooxycyclohex-1-en-1-yl]acetate on the basis of spectroscopic evidence.